Elizabeth R. Wood

PTEN loss confers BRAF inhibitor resistance to melanoma cells through the suppression of BIM expression

This study addresses the role of PTEN loss in intrinsic resistance to the BRAF inhibitor PLX4720. Immunohistochemical staining of a tissue array covering all stages of melanocytic neoplasia (n = 192) revealed PTEN expression to be lost in >10% of all melanoma cases. Although PTEN expression status did not predict for sensitivity to the growth inhibitory effects of PLX4720, it was predictive for apoptosis, with only limited cell death observed in melanomas lacking PTEN expression (PTEN-). Mechanistically, PLX4720 was found to stimulate AKT signaling in the PTEN- but not the PTEN+ cell lines. Liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM) was performed to identify differences in apoptosis signaling between the two cell line groups. PLX4720 treatment significantly increased BIM expression in the PTEN+ (>14-fold) compared with the PTEN- cell lines (four-fold). A role for PTEN in the regulation of PLX4720-mediated BIM expression was confirmed by siRNA knockdown of PTEN and through reintroduction of PTEN into cells that were PTEN-. Further studies showed that siRNA knockdown of BIM significantly blunted the apoptotic response in PTEN+ melanoma cells. Dual treatment of PTEN- cells with PLX4720 and a PI3K inhibitor enhanced BIM expression at both the mRNA and protein level and increased the level of apoptosis through a mechanism involving AKT3 and the activation of FOXO3a. In conclusion, we have shown for the first time that loss of PTEN contributes to intrinsic BRAF inhibitor resistance via the suppression of BIM-mediated apoptosis.

The HSP90 Inhibitor XL888 Overcomes BRAF Inhibitor Resistance Mediated through Diverse Mechanisms

Purpose: The clinical use of BRAF inhibitors is being hampered by the acquisition of drug resistance. This study shows the potential therapeutic use of the HSP90 inhibitor (XL888) in six different models of vemurafenib resistance. Experimental Design: The ability of XL888 to inhibit growth and to induce apoptosis and tumor regression of vemurafenib-resistant melanoma cell lines was shown in vitro and in vivo. A novel mass spectrometry–based pharmacodynamic assay was developed to measure intratumoral HSP70 levels following HSP90 inhibition in melanoma cell lines, xenografts, and melanoma biopsies. Mechanistic studies were carried out to determine the mechanism of XL888-induced apoptosis. Results: XL888 potently inhibited cell growth, induced apoptosis, and prevented the growth of vemurafenib-resistant melanoma cell lines in 3-dimensional cell culture, long-term colony formation assays, and human melanoma mouse xenografts. The reversal of the resistance phenotype was associated with the degradation of PDGFRβ, COT, IGFR1, CRAF, ARAF, S6, cyclin D1, and AKT, which in turn led to the nuclear accumulation of FOXO3a, an increase in BIM (Bcl-2 interacting mediator of cell death) expression, and the downregulation of Mcl-1. In most resistance models, XL888 treatment increased BIM expression, decreased Mcl-1 expression, and induced apoptosis more effectively than dual mitogen-activated protein–extracellular signal–regulated kinase/phosphoinositide 3-kinase (MEK/PI3K) inhibition. Conclusions: HSP90 inhibition may be a highly effective strategy at managing the diverse array of resistance mechanisms being reported to BRAF inhibitors and appears to be more effective at restoring BIM expression and downregulating Mcl-1 expression than combined MEK/PI3K inhibitor therapy. Clin Cancer Res; 18(9); 2502–14. ©2012 AACR.

Evaluating Melanoma Drug Response and Therapeutic Escape with Quantitative Proteomics

The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 μg of total protein). We further showed MEK inhibition to be associated with signaling through the NFκB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor β signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, crenolanib. Together, our studies show LC-MRM to have unique value as a platform for the systems level understanding of the molecular mechanisms of drug response and therapeutic escape. This work provides the proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic.

Inhibition of Wee1, AKT, and CDK4 Underlies the Efficacy of the HSP90 Inhibitor XL888 in an In Vivo Model of NRAS-Mutant Melanoma

The HSP90 inhibitor XL888 is effective at reversing BRAF inhibitor resistance in melanoma, including that mediated through acquired NRAS mutations. The present study has investigated the mechanism of action of XL888 in NRAS-mutant melanoma. Treatment of NRAS-mutant melanoma cell lines with XL888 led to an inhibition of growth, G2–M phase cell-cycle arrest, and the inhibition of cell survival in three-dimensional spheroid and colony formation assays. In vitro, HSP90 inhibition led to the degradation of ARAF, CRAF, Wee1, Chk1, and cdc2 and was associated with decreased mitogen-activated protein kinase (MAPK), AKT, mTOR, and c-jun NH2 kinase (JNK) signaling. Apoptosis induction was associated with increased BIM expression and a decrease in the expression of the prosurvival protein Mcl-1. The critical role of increased BIM and decreased Mcl-1 expression in the survival of NRAS-mutant melanoma cell lines was shown through siRNA knockdown and overexpression studies. In an animal xenograft model of NRAS-mutant melanoma, XL888 treatment led to reduced tumor growth and apoptosis induction. Important differences in the pattern of client degradation were noted between the in vivo and in vitro studies. In vivo, XL888 treatment led to degradation of CDK4 and Wee1 and the inhibition of AKT/S6 signaling with little or no effect observed upon ARAF, CRAF, or MAPK. Blockade of Wee1, using either siRNA knockdown or the inhibitor MK1775, was associated with significant levels of growth inhibition and apoptosis induction. Together, these studies have identified Wee1 as a key target of XL888, suggesting novel therapeutic strategies for NRAS-mutant melanoma. Mol Cancer Ther; 12(6); 901–12. ©2013 AACR.

Fibronectin induction abrogates the BRAF inhibitor response of BRAF V600E/PTEN-null melanoma cells

The mechanisms by which some melanoma cells adapt to Serine/threonine-protein kinase B-Raf (BRAF) inhibitor therapy are incompletely understood. In the present study, we used mass spectrometry-based phosphoproteomics to determine how BRAF inhibition remodeled the signaling network of melanoma cell lines that were BRAF mutant and PTEN null. Short-term BRAF inhibition was associated with marked changes in fibronectin-based adhesion signaling that were PTEN dependent. These effects were recapitulated through BRAF siRNA knockdown and following treatment with chemotherapeutic drugs. Increased fibronectin expression was also observed in mouse xenograft models as well as specimens from melanoma patients undergoing BRAF inhibitor treatment. Analysis of a melanoma tissue microarray showed loss of PTEN expression to predict for a lower overall survival, with a trend for even lower survival being seen when loss of fibronectin was included in the analysis. Mechanistically, the induction of fibronectin limited the responses of these PTEN-null melanoma cell lines to vemurafenib, with enhanced cytotoxicity observed following the knockdown of either fibronectin or its receptor α5β1 integrin. This in turn abrogated the cytotoxic response to BRAF inhibition via increased AKT signaling, which prevented the induction of cell death by maintaining the expression of the pro-survival protein Mcl-1. The protection conveyed by the induction of FN expression could be overcome through combined treatment with a BRAF and PI3K inhibitor.

Quantitative proteomics of breast tumors: Tissue quality assessment to clinical biomarkers

Liquid chromatography‐selected reaction monitoring mass spectrometry (LC‐SRM) is not only a proven tool for clinical chemistry, but also a versatile method to enhance the capability to quantify biomarkers for tumor biology research. As the treatment of cancer continues to evolve, the ability to assess multiple biomarkers to assign cancer phenotypes based on the genetic background and the signaling of the individual tumor becomes paramount to our ability to treat the patient. In breast cancer, the American Society of Clinical Oncology has defined biomarkers for patient assessment to guide selection of therapy: estrogen receptor, progesterone receptor, and the HER2/Neu receptor tyrosine kinase; therefore, these proteins were selected for LC‐SRM assay development. Detailed molecular characterization of these proteins is necessary for patient treatment, so expression and phosphorylation assays have been developed and applied. In addition, other LC‐SRM assays were developed to further evaluate tumor biology (e.g. Ki‐67 for proliferation and vimentin for tumor aggressiveness related to the epithelial‐to‐mesenchymal transition). These measurements combined with biomarkers for tissue quality and histological content are implemented in a three‐tier multiplexed assay platform, which is translated from cell line models into frozen tumor tissues banked from breast cancer patients.

Multiplexed Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry Quantification of Cancer Signaling Proteins

Quantitative evaluation of protein expression across multiple cancer-related signaling pathways (e.g., Wnt/β-catenin, TGF-β, receptor tyrosine kinases (RTK), MAP kinases, NF-κB, and apoptosis) in tumor tissues may enable the development of a molecular profile for each individual tumor that can aid in the selection of appropriate targeted cancer therapies. Here, we describe the development of a broadly applicable protocol to develop and implement quantitative mass spectrometry assays using cell line models and frozen tissue specimens from colon cancer patients. Cell lines are used to develop peptide-based assays for protein quantification, which are incorporated into a method based on SDS-PAGE protein fractionation, in-gel digestion, and liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM/MS). This analytical platform is then applied to frozen tumor tissues. This protocol can be broadly applied to the study of human disease using multiplexed LC-MRM assays.

Abstract 2491: Quantification of biomarker expression, phosphorylation, and mutation in cancer using TMT labeling prior to liquid chromatography-multiple reaction monitoring mass spectrometry

Introduction: Quantitative mass spectrometry assays using the Tandem Mass Tag (TMT) labeling strategy have been developed for measuring biologically relevant phosphorylation and mutation sites, which are not easily amenable to quantification in standard proteomics workflows. Quantification of the c-terminal peptide from HER2, which can be phosphorylated at Tyrosine 1248, reports on potential binding of Shc and activation of Ras signaling. In melanoma, N-Ras mutations at Q61 can occur de novo or develop as a resistance mechanism to B-Raf inhibition. However, tryptic peptides containing these specific sites have poor or no signal for liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) quantification. Different proteolysis strategies and TMT labeling enhanced the signal intensity of those specific peptides and enabled quantification of these candidate biomarkers. The quantification of target peptides with specific phosphorylation and mutation sites will help to understand cancer biology and may predict drug response to support individualized selection of therapy. Experimental Procedures: Model systems included breast cancer (MCF7, BT474) and melanoma (N-Ras WT M257, N-Ras Q61K M245, N-Ras Q61R 2032, N-Ras Q61L M318) cell lines inter al. Peptide-based quantitative assays were developed for HER2 expression and phosphorylation as well as N-Ras expression and mutation using TMT labeling prior to LC-MRM analysis. Endogenous peptides including wild type N-Ras and all known Q61 mutations have been synthesized and tested for TMT labeling efficiency with LC-MRM. Then, proteins were fractionated by SDS-PAGE from whole cell lysates. Different proteolytic strategies including chymotrypsin or Glu-C digestion were optimized for biomarker quantification. The assay characteristics were evaluated using serial dilutions of cell lysate before analysis of clinical specimens. Data Summary: LC-MRM assays for both unmodified and phosphorylated HER2 as well as wild type and mutant N-Ras have been developed and characterized using cell line models. TMT labeling efficiency has been evaluated under different conditions to accommodate different digestion buffers. Assays have been implemented to study these biomarkers. Conclusions: Proteomic quantification of cancer biomarkers defined by known biology, including expression, phosphorylation, and mutation status, has the potential to improve the selection of targeted therapeutics and prediction of drug response. Citation Format: Yi Chen, David J. Britton, Kim Paraiso, Inna Fedorenko, Elizabeth R. Wood, Anthony Magliocco, Vernon K. Sondak, Keiran Smalley, Ian Pike, John M. Koomen. Quantification of biomarker expression, phosphorylation, and mutation in cancer using TMT labeling prior to liquid chromatography-multiple reaction monitoring mass spectrometry. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2491. doi:10.1158/1538-7445.AM2014-2491

Abstract 5112: Proteomic investigation of melphalan resistance in multiple myeloma to support selection of combination therapy

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Proteomic analyses using iTRAQ and reaction monitoring mass spectrometry are used to examine the mechanism of melphalan resistance and build an assessment platform to enable future selection of combination therapy in patients. Experimental Procedures: Drug IC50 values and interactions are assessed using cell viability measurements. Protein expression analysis using isobaric tags for relative and absolute quantification (iTRAQ) was used to compare melphalan resistant cell lines (8226/LR5 and U266/LR6) to their isogenic, naive counterparts (RPMI-8226, U266). In this study, iTRAQ was used to identify proteins that have changed in expression level during the development of melphalan resistance in multiple myeloma (MM). In addition to manual selection of differentially expressed proteins, the resulting quantitative data were coalesced into pathway models using Metacore, GeneGO for further analysis. For targeted protein detection and quantification, liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM) assays are developed and implemented. Data Summary: Melphalan resistant cells are more susceptible to other chemotherapy agents, including steroids, proteasome inhibitors, and geldanamycin derivatives, than their isogenic, naive counterparts. To investigate the differences, iTRAQ showed numerous changes in protein expression, including redox stress components, mitochondrial proteins, and heat shock proteins (HSPs). LC-MRM assays were developed for proteins detected in the iTRAQ experiments that are current chemotherapy targets, e.g. HSPs and proteasome components. Furthermore, assays have also been developed for additional protein targets of other chemotherapy agents, e.g. topoisomerases and the glucocorticoid receptor, that were not detected in the iTRAQ results. LC-MRM was then used to examine the expression of these proteins in the four model cell lines listed above and in the naive cells following the onset of drug treatment. Endogenous proteins were quantified in less than 10,000 cells. These assays can be combined to develop an assessment tool to help elucidate the mechanisms of drug resistance and enable the assessment of myeloma cells from patients’ bone marrow aspirates. Conclusions: Quantification of the proteomes of myeloma cell lines using iTRAQ provided insights into the mechanism of melphalan resistance in multiple myeloma. Investigation of the expression of these proteins and other drug targets in myeloma cell lines has produced an LC-MRM platform that can be translated for patient assessment to assist in selection of chemotherapy combinations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5112. doi:10.1158/1538-7445.AM2011-5112

Quantification of Breast Cancer Protein Biomarkers at Different Expression Levels in Human Tumors

Liquid chromatography-selected reaction monitoring (LC-SRM) mass spectrometry has developed into a versatile tool for quantification of proteins with a wide range of applications in basic science, translational research, and clinical patient assessment. This strategy uniquely complements traditional pathology approaches, like hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). The multiplexing capabilities offered by mass spectrometry are currently unmatched by other techniques. However, quantification of biomarkers in tissue specimens without the other data obtained from H&E-stained slides or IHC, including tumor cellularity or percentage of positively stained cells inter alia, may not provide as much information that is needed to fully understand tumor biology or properly assess the patient. Therefore, additional characterization of the tissue proteome is needed, which in turn requires the ability to assess protein markers across a wide range of expression levels from a single sample. This protocol provides an example of multiplexed analysis in breast tumor tissue quantifying specific biomarkers, specifically estrogen receptor, progesterone receptor, and the HER2 receptor tyrosine kinase, in combination with other proteins that can report on tissue content and other aspects of tumor biology.